Mechanisms of Autophagy and Proteasomal Degradation in Targetting Disease Aggregates: An In Situ Cryo-Electron Tomography Approach

Neurodegenerative diseases (ND) characterized by progressive and fatal motor and cognitive decline, include Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis/Frontotemporal Dementia (ALS/FTD), and many others. In HD, the huntingtin (Htt) protein undergoes a liquid-to-solid phase transition, forming amyloid-like fibrils through soluble and amorphous intermediates. Mammalian cells, including neurons, rely on two major pathways -- autophagy and ubiquitin-proteasome degradation (UPS) -- to clear misfolded and aggregated proteins. My previous work, using in situ cryo-electron tomography (cryo-ET), revealed that long polyQ repeats form a fibrillar core, surrounded by amorphous intermediates, which compared to the fibrils, are more readily taken up by phagophores and autophagosomes typical of macroautophagy (Zhao, et al., Mol Cell 2024). This work highlights the unique power of cryo-ET in bridging cell and structural biology, particularly in addressing key questions in the field of autophagy. Currently, I aim to understand how the UPS interacts with different polyQ phases at the molecular level, using in situ cryo-ET and subtomogram averaging. The analyses reveal that the chaperone AAA+ ATPase VCP facilitates the clearance of polyQ intermediates by interacting not only with 26S proteasomes but also with 20S proteasomes through a distinct coupling mechanism, likely mediated by VCP’s C-terminal hydrophobic-tyrosine-X (HbYX) motif, which binds directly to the 20S proteasome. These findings raise critical mechanistic questions that require urgent investigation. This research proposal aims to integrate cryo-ET with biophysical, biochemical, and cellular approaches to address fundamental questions in protein quality control pathways with far-reaching therapeutic implications for ND. Research questions: (1) Structural and Molecular Mechanisms of CMA Chaperone-mediated autophagy (CMA) is a critical selective autophagic pathway for the clearance of misfolded and aggregated proteins implicated in ND; however, its structural basis remains completely unknown. This study will provide a concrete structural elucidation of CMA, its cross-talk with macroautophagy, and its impact on lysosomal integrity, with far-reaching implications for therapeutic strategies. Key outcomes: 1. Determining the atomic structure of the Lamp2A translocon complex in vitro -- a key CMA component responsible for directly importing misfolded proteins into the lysosome for degradation 2. In situ identification of Lamp2A oligomerization states, their impact on lysosomal architecture, and the recruitment of CMA machineries in protein unfolding. 3. Elucidation of the roles of Lamp (lysosome-associated membrane protein) homologs Lamp1, 2B, 2C in autophagy -- involvement in autophagosome-lysosome fusion in situ. 4. Determining structurally how disease aggregates block CMA and impair lysosomal health in situ. (2) VCP-20S Proteasome Coupling The UPS is essential for cellular proteostasis, yet past research has focused on the 26S proteasome, leaving the 20S proteasome and its alternative gating mechanisms under-explored. This study will be distinct in establishing a novel mechanistic framework for VCP-20S coupling in UPS, with profound implications for therapeutic strategies for ND. Key outcomes include: 1. Identification of key structural elements that mediate the coupling mechanism. 2. A detailed understanding of VCP-20S coupled substrate degradation in vitro and in the cell. 3. Insights into the generality of the VCP-20S coupling across different aggregate models including HD and AD, and in the mouse neuronal systems. By elucidating these mechanisms, I aim to re-define the structural and mechanistic basis of major protein quality control pathways critical for clearing misfolded and aggregated proteins, with profound therapeutic implications for ND.